Vitamin A prevents lipopolysaccharide-induced injury on tight junctions in mice.

Abstract Vitamin A (VA) is one of the most widely used food supplements, but its molecular mechanisms largely remain elusive. Previously, we have demonstrated that VA inhibits the action of lipopolysaccharide (LPS) on intestinal epithelial barrier function and tight junction proteins using IPEC‐J2 cells, one of representative intestinal cell lines as a cellular model. These exciting findings stimulated us continue to determine the effects of VA on LPS‐induced damage of intestinal integrity in mice. Our results demonstrated that LPS treatment caused reductions of the mRNA levels of tight junction proteins including Zo‐1, Occludin, and Claudin‐1, well‐known biomarkers of intestinal integrity, and these reductions were reversed by VA pretreatment. Intestinal immunofluorescent results of Claudin‐1 revealed that LPS disrupted the structure of tight junction and reduced the expression of Claudin‐1 at protein level, which was reversed by VA pretreatment. These results suggest that VA may exert a profound role on preventing intestinal inflammation in vivo.


| INTRODUC TI ON
Vitamin A (VA) is a commonly used food supplement that maintains visual function, tissue development, differentiation, and immune response. Particularly, VA has been confirmed having its critical role of anti-inflammatory effect Zhang et al., 2019).
Interestingly, VA plays an essential role in tight junctions in vitro (Osanai, 2017;Rybakovsky et al., 2017). Tight junctions are important components of the intestinal epithelial barrier. The development of most diseases is related to the intestinal epithelial barrier in the clinic, and the intestine has a function of separating the substances in the intestinal lumen and preventing the invasion of pathogenic antigens (Buckley & Turner, 2018;De Santis, Cavalcanti, Mastronardi, Jirillo, & Chieppa, 2015). Thus, alteration of tight junctions is becoming an important biomarker of determining the function of intestine (Wardill, Gibson, Logan, & Bowen, 2014). Studies have confirmed that LPS, one commonly and important inflammation activator, produces damages on tight junctions in vitro (Chen et al., 2015;Main, Weber, Baumgard, & Gabler, 2012). Previously, we have shown that VA protected against LPS-induced damage on tight junctions in vitro (He et al., 2019). However, whether or how effect of VA on tight junction of intestine in vivo remains elusive.

Abstract
Vitamin A (VA) is one of the most widely used food supplements, but its molecular mechanisms largely remain elusive. Previously, we have demonstrated that VA inhibits the action of lipopolysaccharide (LPS) on intestinal epithelial barrier function and tight junction proteins using IPEC-J2 cells, one of representative intestinal cell lines as a cellular model. These exciting findings stimulated us continue to determine the effects of VA on LPS-induced damage of intestinal integrity in mice. Our results demonstrated that LPS treatment caused reductions of the mRNA levels of tight junction proteins including Zo-1, Occludin, and Claudin-1, well-known biomarkers of intestinal integrity, and these reductions were reversed by VA pretreatment. Intestinal immunofluorescent results of Claudin-1 revealed that LPS disrupted the structure of tight junction and reduced the expression of Claudin-1 at protein level, which was reversed by VA pretreatment. These results suggest that VA may exert a profound role on preventing intestinal inflammation in vivo.

K E Y W O R D S
inflammation, intestine, tight junctions, vitamin A Thus, we use miniature mice to further explore the effect of VA on tight junctions in vivo.

| Materials
Vitamin A was purchased from Sigma-Aldrich and dissolved in DMSO to prepare a stock solution of 100 mM. LPS was purchased from Sigma-Aldrich as well and dissolved in PBS to prepare the solution stored at −20°C. IL-6, TNF-α, and β-actin were purchased from Cell Signaling Technology. Sodium carboxymethyl cellulose was purchased from Aladdin Biochemical Technology Co., Ltd. The mice were treated with oral VA for 4 days and intraperitoneal LPS for 24 hr. The doses of LPS and VA were adopted from previous published work (Kim, Kim, Park, Kim, & Chang, 2015).

| Collection of mice intestinal tissue samples
At the end of each experiment, the mouse cervical spine was dislocated and the intestinal tissues were collected immediately. After washing with PBS at 4°C, and intestinal tissues were either for cryosections or stored in liquid nitrogen for further study.

| Reverse transcriptase-polymerase chain reaction
The PCR system consisted of 5.0 μl of YBR Green qPCR Mix, 0.2 μl of cDNA, 0.3 μl of each primer, and 4.2 μl of double-distilled water in a final volume of 20 μl. The detailed information of each primer was presented in Table 1. Each sample was determined in triplicate, and the housekeeping gene GAPDH was used as the internal standard for the PCR. Quantitative real-time PCR was performed with a Real Master Mix SYBP ROX (5 Prime) according to the manufacturer's protocols using the same pig GAPDH primer.

| Western blot
Tissues proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to membranes, and then incubated overnight at 4°C with different primary antibodies described in Reagents section above (Cell Signaling Technology) in buffer containing bovine serum albumin. Membranes were washed with triethanolamine-buffered saline (TBS) containing 0.05% Tween-20, blotted with secondary antibody for 1 hr at room temperature, and then washed again three times. Pierce Super Signal chemiluminescent substrate (Rockford, IL, USA) was added, and the blot was imaged immediately on a ChemiDoc system (Tanon 4600) and a Perfection V500 camera (Epson). Band intensities were quantified using ImageJ.

| Preparation of cryosections
Each intestine tissue was transferred from fixative to 300 μl 2.3 M sucrose in 0.1 M sodium phosphate pH 7.4 at 4°C for 1 hr, then embedded and quick-frozen in OCT compound (Tissue-Tek). 5-μm cryosections were cut at −20°C collected on chrome alum/gelatinsubbed slides, and stored up to 24 hr at −20°C. Optimally oriented cryosections were selected for further processing.

| Statistical analysis
SPSS16.0 (SPSS Inc.) was used for statistical analysis. Measurement data were presented as mean ± standard deviation (SD). Comparison of means among multiple groups was performed by one-way analysis of variance (ANOVA), followed Dunnett post hoc test. A statistical significance was defined when p < .05.

| Effect of vitamin A on gene expression of tight junction in intestinal tissues in mice
We utilized intestine tissues among four groups to determine whether there is any alteration of tight junctions. mRNA levels of Zo-1, Occludin, and Claudin-1 were determined by Reverse transcriptase-polymerase chain reaction (RT-PCR). As shown in Figure

| Effect of Vitamin A on inflammatory responses in mice
To explore anti-inflammatory function of VA on intestinal tissues, alterations of TNF-α and IL-6 as biomarkers of inflammation during

| Immunofluorescent evaluation of the localization and expression of tight junction
Immunofluorescence was used to detect the localization and expression of tight junctions since results collected by immunofluorescence microscopy are more intuitive. As shown in Figure 3, tight junction of Claudin-1 is neatly arranged in mouse intestine tissues in the control group, while VA alone profoundly enhanced Claudin-1 expression with perfect arrangement. However, LPStreated group showed severely structural disruption with decrease in tight junction protein Claudin-1. In contrast, VA treatment protected this disruption when the mice were treated with both VA and LPS.   (Huang et al., 2015). Interestingly, Sterlin et al. (2019) reported that human gut microbiomes evolved in the absence of immunoglobulin A (lgA).

| D ISCUSS I ON
Furthermore, Ma et al. (2018) summarized that intestinal bacteria-immune crosstalk and nutritional regulation on their interplay, to understanding their interactions. In contrast, another report does not support that a short-term pro-inflammatory effect of A. muciniphila strain in the IL-10 −/− mouse model for IBD (Ring et al., 2019). Another study reveals that RegIIIb-target recognition, killing of Gram-negative bacteria in infectious diarrhea, proposes avenues toward novel therapeutic interventions for Salmonella diarrhea (Miki, Okada, & Hardt, 2018). Using human patient specimens, Kiely, Pavli, and O'Brien (2018) demonstrated that inflammation is related to microbiome, which is spatially and timely altered in inflammatory bowel disease (IBD). Interestingly, Mamantopoulos, Ronchi, McCoy, and Wullaert (2018) analyzed the association between maternal inheritance and long-term separate housing and host-microbiota interactions, illustrating the importance of host-microbiota and inflammatory responses. As summarized in Figure 4, we find that VA enhances expressions

| CON CLUS IONS
The present study reveals that VA enhances expression of intestinal tight junction proteins and reverses both LPS-induced increase in inflammation and decrease in intestinal tight junction proteins. These results for the first time confirm that VA plays a profound role on preventing intestinal inflammation.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.